The retinoblastoma gene, Rb, is a prototype growth suppressor gene the inactivation of which is associated with tumor development. The fact that the Rb protein (pRb) can be inactivated by binding to the oncoproteins of some DNA tumor viruses suggest the existence of functionally homologous cellular proteins. In our attempt to isolate such protjeins, we made the surprising discovery that pRb shares an antigenically homologous domain, P16, with the SV40 large T antigen (LT). Comparison of the protein sequence between pRb and LT revealed the existence of other homology domains. The mutation of these domains critically affected their functions. Mutations at the homology regions in pRb inactivate its growth suppression function and its binding to E2F- 1 and LT. While mutations at the homology regions of LT have no effect on its binding to either p53 or pRb, its binding to the B subunit of DNA polymerase alpha and therefore its ability to initiate DNA replication was completely abolished. Consistent with this observation is the fact that almost all temperature-sensitive LT mutants (which affect the initiation of DNA replication) are mutated at the homology regions. Inspection of the structure of subunits A and B revealed the presence of the LXCXE motif known to be responsible for the binding to the pocket regions of pRb-like proteins. We concluded that the homology regions represent part of a structural motif (pocket region) needed for the binding to the structural motif LXCXE. We hypothesize that proteins with a pocket region such as pRb and p107, may regulate the initiation of DNA replication by interfering with the binding of the pocket regions of LT or LT-like proteins to the LXCXE region of DNA polymerase alpha A and/or B. In turn, proteins with a LXCXE domain, such as E1a may modulate DNA replication by binding to the pocket of Rb or Rb-like proteins. Experiments are proposed to examine the role of the binding of the pocket regions of Rb and Rb-like proteins to the LXCXE domains of LT, DNA polymerase alpha subunits A or B on the known inhibitory effect of Rb on the Lt dependent replication process. Since the antibodies RB1-AB 16-2 recognizes the p16 region, we surmise that it may also recognize Rb-like and LT-like proteins that possess the same region. Sequence analysis of the first three clones, isolated from gt11 libraries with RB1-Ab16-2, revealed striking homology between one of the clones with LT. We have tentatively named this clone CHLA-1 (Cellular Homologue of the SV40 Large T Antigen) to denote the fact that there are at least three regions of structural homology between them. They are the p9 Rb/LT homology region, the p16 Rb/LT homology region and the LXCXE domain. Experiments are proposed to characterize CHLA-1 with respect to its temporal and spatial expression pattern, its DNA binding sequence and the proteins that it can bind to.